1. Field of the Invention
This invention relates to the field of Peptide Nucleic Acid (PNA) synthesis. More particularly, this invention relates to PNA synthons suitable for the synthesis and deprotection of PNA-DNA chimeras.
2. Description of the Background Art
Peptide Nucleic Acids (PNAs) are synthetic polyamides which are promising candidates for the sequence-specific regulation of DNA expression and for the preparation of gene targeted drugs. See European Patent applications EP 92/01219 and 92/01220 which are herein incorporated by reference. PNAs are biopolymer hybrids which possess a peptide-like backbone to which the nucleobases of DNA are attached. Specifically, PNAs are synthetic polyamides comprised of repeating units of the amino acid, N-(2-aminoethyl)-glycine, to which the nucleobases adenine, cytosine, guanine, thymine and uracil are attached through a methylene carbonyl group. Other natural and unnatural nucleobases, such as pseudo isocytosine, 5-methyl cytosine, pseudouracil, isocytosine, hypoxanthine and 2,6-diaminopurine, among many others, also can be incorporated in PNA synthons (see FIG. 8).
PNAs are now routinely synthesized from monomers (PNA synthons) protected according to the t-Boc/benzyl protection strategy, wherein the backbone amino group of the growing polymer is protected with the t-butyloxycarbonyl (t-Boc) group and the exocyclic amino groups of the nucleobases, if present, are protected with the benzyloxycarbonyl (benzyl) group. PNA synthons protected using the t-Boc/benzyl strategy are now commercially available but are inconvenient to use because, among other reasons, harsh acidic conditions are required to remove these protecting groups.
The t-Boc/benzyl protection strategy requires very strong acids to remove all of the benzyl side chain nucleobase protecting groups. Typically, PNA oligomers are exposed to hydrofluoric acid or trifluoromethane sulfonic acid for periods of time often exceeding one hour to completely remove the benzyl side chain protecting groups. This harsh acid treatment needed for final deprotection will often decompose, among other acid sensitive moieties, nucleic acids and carbohydrates which might be attached to the nucleic acid oligomer. Furthermore, the use of hazardous acids such as hydrofluoric acid or trifluoromethane sulfonic acid is not commercially embraced in view of safety concerns for the operators and the corrosive effect on automation equipment and lines. The above described harsh conditions are particularly unsuitable for the synthesis of nucleic acids since the strong acid deprotection conditions will at least partially decompose nucleic acids.
In addition, the t-Boc/benzyl protection strategy is not orthogonal but differential. A differential strategy is defined as a system of protecting groups wherein the protecting groups are removed by essentially the same type of reagent or condition, but rely on the different relative rates of reaction to remove one group over the other. For example, in the t-Boc/benzyl protecting strategy, both protecting groups are acid labile, with benzyl groups requiring a stronger acid for efficient removal. When acid is used to completely remove the more acid labile t-Boc protecting groups, there is a potential that a percentage of benzyl groups will also be removed contemporaneously. Specifically, the t-Boc protecting group must be removed from the amino group backbone during each synthetic cycle so the next monomer can be attached to the backbone at the free amino site thereby allowing the polymeric chain to grow. The deprotection of the t-Boc amino protected backbone is accomplished using a strong acid such as trifluoroacetic acid. During this deprotection and subsequent construction of the PNA oligomer, removal of the nucleobase side chain protecting groups, i.e., the benzyls, is undesirable. However, trifluoroacetic acid is potentially strong enough to prematurely deprotect a percentage of the side chain benzyl groups, thereby introducing the possibility of polymer branching and reducing the overall yield of desired product.
An orthogonal strategy, on the other hand, removes the protecting groups under mutually exclusive conditions, e.g., one group is removed with acid while the other group is removed with base. Breipohl et al. have described an orthogonally protected PNA synthon using 9-fluorenylmethyloxycarbonyl (Fmoc) as the backbone protecting group and a triphenylmethyl (trityl) group as the side chain nucleobase protecting group. Breipohl et al. 1st Australian Peptide Conference, Great Barrier Reef, Australia, Oct. 16-21, 1994. This protection methodology, however, is incompatible with standard nucleic acid synthesis methodology.
Christensen et al. have described orthogonal PNA synthons wherein the t-Boc amino backbone protecting group is removed in strong acid then reprotected with 9-fluorenylmethyloxycarbonyl (Fmoc), a base labile protecting group. Christensen, L. et al. "Innovation and Perspectives in Solid Phase Synthesis and Complementary Technologies-Biological and Biomedical Applications," 3rd SPS Oxford Symposia (1994). Although this protection strategy eliminates the potential for premature deprotection of the exocyclic amino group of the side chain nucleobase, extra steps are involved in preparation of this monomer. Additionally, strong acids such as hydrofluoric acid or trifluoromethane sulfonic acid still are required to remove the benzyl side chain protecting groups.
Nucleic acids (DNA and RNA) are now routinely synthesized using automated machines, numerous synthesis supports and various protection chemistries. The following U.S. patents cover a broad range of differing supports and protection chemistries and are herein incorporated by reference. U.S. Pat. Nos. 5,262,530; 4,415,732; 4,458,066; 4,725,677 (RE 34,069); and 4,923,901. Automated equipment and reagents are commercially available from PerSeptive Biosystems, Perkin Elmer (Applied Biosystems Division) and Pharmacia. Special 5'-amino synthons are described in Smith et al., Nucleic Acids Res. (1985) 13:2399 and in Sproat et al., Nucleic Acids Res. (1987) 15:6181. Special 5'-thio synthons are described in Sproat et al., Nucleic Acids Res. (1987) 15:4837. The reagents described in the above references are suitable for use on standard DNA synthesis instruments.
The preferred commercial method for nucleic acid synthesis utilizes the above reagents and methods as generally described by Koester et al. in U.S. Pat. No. 4,725,677 (RE 34,069). Consequently, the preferred synthons are .beta.-cyanoethyl phosphoramidites having acid labile protection of the backbone 5' hydroxyl group and base labile acyl-type protection of the exocyclic amino groups of the nucleobases.
The preferred acid labile backbone protecting group is 4,4'-dimethoxytriphenylmethyl (DMT). DMT is typically chosen because it can be removed farily rapidly (1-3 mintues) during each synthetic cycle with solutions containing 1-4% dichloroacetic acid or trichloroacetic acid in dichloromethane. Protecting groups with increased acid lability compared to DMT are susceptible to premature deprotection during the acid catalyzed coupling reactions (tetrazole is typically the acidic species). Protecting groups with decreased acid lability compared to DMT require longer reaction times and/or harsher reaction conditions for complete removal. Generally, harsher acidic deprotection conditions are avoided since the purine nucleobases are particularly susceptible to decomposition in acid. Although the aforementioned problems with protecting groups and synthetic conditions may be minimal during each synthetic cycle, the cumulative effect can generate significant impurities in oligonucleotide synthesis. Accordingly, as the length of the oligonucleotide increases, its purity tends to decrease.
Generally, base labile protecting groups are utilized for protection of the exocyclic amino groups of the nucleobases so that an orthogonally protected nucleic acid synthon results. The base labile protecting groups typically remain a part of the growing nucleic acid chain, then are removed simultaneously with the cleavage of synthesized nucleic acid from the solid support. A concentrated ammonium hydroxide solution is often used for the "deprotection and cleavage step." Koester et al. used base labile acyl-type protecting groups which are usually treated for 6-24 hours at elevated temperature (about 55.degree. C.) for complete removal of these nucleobase protecting groups. Other protecting groups have been developed which are removed under the same conditions but in less time (from about 15-60 minutes). Examples of these improved protecting groups include phenoxyacetyl, t-butyl phenoxyacetyl and amindine-type protecting groups. While these protecting groups have increased base lability, typically only the time necessary for removal is reduced.
Following from the above discussion, PNA synthons suitable for the construction of various nucleic acid oligomers should be compatible with current DNA syntheic methodologies so existing techniques and equipment can be utilized. Specifically, an appropriately acid labile protecting group for backbone protection and an appropriate base labile protecting group for nucleobase protection need to be incorporated into the design of a PNA synthon. The ability to syntheisze PNA synthons to meet the above requirements would allow various combinations of nucleic acids to be routinely synthesized and, consequently, allow the expansion of scientific investigations into the utility of these complex molecules.
Another current limitation on the synthesis of PNA synthons is the formation of the side chain nucleobase protecting group. Generally, the exocyclic amino groups of the nucleobases, e.g., cytosine, adenine, and guanine, are protected as carbamates via reaction with activated carbonates or chloroformates. This method of carbamate formation suffers from the disadvantage that many chloroformates are unstable or that the chloroformates are not appreciably reactive with the mildly nucleophilic exocyclic amino groups of the nucleobases. Other methods of carbamate formation used for nucleobases include the use of imidazolides and alkyl imidazolium salts as acylating agents. See Watkins et al, J. Org. Chem., (1982) 47:4471-77 and Watkins et al., J. Am. Chem. Soc., (1982) 104:5702-08. While imidazolides and alkylated imidazolides appear to overcome some of the difficulties associated with carbamate formation, their widespread use with nucleobases has yet to be reported. Recently, the 4-methoxy-triphenylmethyl (MMT) group was presented as another exocyclic amino protecting group for PNA synthon side chain nucleobases. Breipohl et al. 1st Australian Peptide Conference, Great Barrier Reef, Australia, Oct. 16-21, 1994. The MMT group, however, is not a carbamate protecting group.
In addition to the above, the synthesis of a selectively protected guanine PNA synthon has been elusive. The reported guanine PNA synthons are protected as O-6-benzyl ethers but optionally possess benzyl protection of the exocyclic 2-amino group. See European Patent Application EP 92/01219 and U.S. patent applications PCT/US92/10921. Given the relative reactivity of the 6-carbonyl group (enol form) and the more reactive exocyclic 2-amino group, there is no compelling reason for protecting the 6-carbonyl group during PNA synthesis, whereas protection of the more reactive 2-amino group is preferred.
The benzyloxycarbonyl group has been utilized in DNA synthesis for the protection of the exocyclic amino groups of the nucleobases cytosine, adenine and guanine. See Watkins et al, J. Org. Chem., (1982) 47:4471-77 and Watkins et al., J. Am. Chem. Soc., (1982) 104:5702-08. Nonetheless, the guanine synthon was difficult to prepare because the exocyclic 2-amino group of guanine was not reactive toward reagents routinely used to introduce the benzyl group, such as benzyl chloroformate, benzyloxycarbonyl imidazolide and N-alkylated benzyloxycarbonyl imidazole. Consequently, a non-conventional multi-step procedure was described wherein treatment with phenyl chlorothioformate simultaneously protected both the 6-carbonyl group and the exocyclic 2-amino group. Thereafter, the adduct was converted to a carbamate protected guanine compound whereby the 6-carbonyl protecting group was subsequently removed. Nonetheless, this indirect method is laborious because it requires the formation of a carbamate protecting group from the initial adduct and the subsequent deprotection of the 6-carbonyl group.
Suitably protected derivatives of 2-amino-6-chloropurine may be converted to guanine compounds by displacement of the 6-chloro group with oxygen nucleophiles. See Robins et al, J. Am. Chem. Soc. (1965) 87:4934, Reese et al., Nucl. Acids Res., (1981) 9: 4611 and Hodge et al., J. Org. Chem., (1990) 56:1553. Indeed, suitably protected derivatives of 2-amino-6-chloropurine are the starting materials currently described for preparation of the reported guanine PNA synthons. See European Patent Application EP 92/ 01219 and U.S. patent application PCT/US 92/10921.
The inventors of PNA describe a guanine synthon having no protection of the exocyclic 2-amino group but having the 6-carbonyl group protected as a benzyl ether. See European Patent Application EP 92/01219. This protection strategy is surprising because the more reactive 2-amino group will likely react (at least marginally) with the activated carboxylic acid group of other PNA monomers, thereby causing branching of the synthesized polymer. Conversely, the enol, which exists when the 6-carbonyl group remains unprotected, is not reactive enough to result in polymer branching and therefore should require no protection. This particular approach is inconsistent with t-Boc/benzyl protection strategy they employed for the other PNA synthons.
In a more recent patent application, the guanine PNA synthon has both benzyl protection of the exocyclic 2-amino group and a 6-carbonyl group protected as a benzyl ether. See U.S. patent application PCT/US 92/10921. As previously discussed, there is no compelling rationale for protecting the 6-carbonyl group of the guanine PNA synthon. However, protection of the 6-carbonyl group enables selective ionization of the exocyclic 2-amino group of the guanine heterocycle thereby facilitating the reaction of the ionized 2-amino group with conventional benzyl protecting reagents (e.g. benzyloxycarbonyl imidazole). Nonetheless, protection of the exocyclic 2-amino group occurs on a guanine derivative additionally protected at the 6-carbonyl group of the nucleobase. Thus, the resulting synthon has both exocyclic 2-amino group and 6-carbonyl group protection. Hence, there remains no reported convenient high yield synthesis of a guanine PNA synthon having selective carbamate protection of the exocyclic 2-amino group, wherein the 6-carbonyl group remains unprotected.
Solid phase peptide synthesis methodology is applicable to the synthesis of PNA oligomers, but often requires the use of harsh reaction conditions unsuitable for DNA synthesis, and consequently, PNA-DNA chimera synthesis too. In the above-mentioned t-Boc/benzyl protection scheme, the final deprotection of side-chains and release of the PNA molecule from the solid support is most often carried out by the use of strong acids such as anhydrous hydrofluoric acid (HF) (Sakakibara, et al., Bull. Chem. Soc. Jpn., 1965, 38, 4921), boron tris (trifluoroacetate) (Pless, et al., Helv. Chim. Acta, 1973, 46, 1609), and sulfonic acids such as trifluoromethanesulfonic acid and methanesulfonic acid (Yajima, et al., J. Chem. Soc., Chem. Comm., 1974, 107). This conventional strong acid (e.g., anhydrous HF) deprotection method, produces very reactive carbocations that may lead to alkylation and acylation of sensitive residues in the PNA chain. Such side-reactions are only partly avoided by the presence of scavengers such as anisole, phenol, dimethyl sulfide, and mercaptoethanol. Thus, the sulfide-assisted acidolytic S.sub.N 2 deprotection method (Tam, et al., J. Am. Chem. Soc., 1983, 105, 6442 and J. Am. Chem. Soc., 1986, 108, 5242), the so-called "low," which removes the precursors of harmful carbocations to form inert sulfonium salts, is frequently employed in peptide and PNA synthesis, either solely or in combination with "high" methods. Less frequently, in special cases, other methods used for deprotection and/or final cleavage of the PNA-solid support bond are, for example, such methods as base-catalyzed alcoholysis (Barton, et al., J. Am. Chem. Soc., 1973, 95, 4501), and ammonolysis as well as hydrazinolysis (Bodanszky, et al., Chem., Ind., 1964, 1423), hydrogenolysis (Jones, Tetrahedron Lett., 1977, 2853 and Schlatter, et al., Tetrahedron Lett., 1977, 2861) ), and photolysis (Rich and Gurwara, J. Am. Chem. Soc., 1975, 97, 1575)).
Based on the recognition that most operations are identical in the synthetic cycles of solid-phase peptide synthesis (as is also the case for solid-phase PNA), a new matrix, PEPS, was recently introduced (Berg, et al., J. Am. Chem. Soc., 1989, 111, 8024 and International Patent Application WO 90/02749) to facilitate the preparation of large numbers of peptides. This matrix is comprised of a polyethylene (PE) film with pendant long-chain polystyrene (PS) grafts (molecular weight on the order of 10.sup.6). The loading capacity of the film is as high as that of a beaded matrix, but PEPS has the additional flexibility to suit multiple syntheses simultaneously.
Two other methods proposed for the simultaneous synthesis of large numbers of peptides also apply to the preparation of multiple, different PNA molecules. The first of these methods (Geysen, et al., Proc. Natl. Acad. Sci. USA, 1984, 81, 3998) utilizes acrylic acid-grafted polyethylene-rods and 96-microtiter wells to immobilize the growing peptide chains and to perform the compartmentalized synthesis. While highly effective, the method is only applicable on a microgram scale. The second method (Houghten, Proc. Natl. Acad. Sci. USA, 1984, 82, 5131) utilizes a "tea bag" containing traditionally-used polymer beads. Other relevant proposals for multiple peptide or PNA synthesis include the simultaneous use of two different supports with different densities (Tregear, in "Chemistry and Biology of Peptides," J. Meienhofer, ed., Ann Arbor Sci., Publ., Ann Arbor, 1972, pp. 175-178), combining of reaction vessels via a manifold (Gordman, Anal. Biochem., 1984, 136, 397), multicolumn solid-phase synthesis (e.g. Krchnak, et al., Int. J. Peptide Protein Res., 1989, 33, 209), and Holm and Meldal, in "Proceedings of the 20th European Peptide Symposium," G. Jung and E. Bayer, eds., Walter de Gruyter & Co., Berlin, 1989, pp. 208-210), and the use of cellulose paper (Eichler, et al., Collect. Czech. Chem. Commun., 1989, 54, 1746).
While the conventional cross-linked styrene/divinylbenzene copolymer matrix and the PEPS supports are presently preferred in the context of solid-phase PNA synthesis, a nonlimiting list of examples of solid supports which may be of relevance are: (1) Particles based upon copolymers of dimethylacrylamide cross-linked with N,N'-bisacryloylethylenediamine, including a known amount of N-tertbutoxycarbonyl-beta-alanyl N'-acryloylhexamethylenediamine. Several spacer molecules are typically added via the beta alanyl group, followed thereafter by the amino acid residue moietys. Also, the beta alanyl-containing monomer can be replaced with an acryloyl sarcosine monomer during polymerization to form resin beads. The polymerization is followed by reaction of the beads with ethylenediamine to form resin particles that contain primary amines as the covalently linked functionality. The polyacrylamide-based supports are relatively more hydrophilic than are the polystyrene-based supports and are usually used with polar aprotic solvents including dimethylformamide, dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, et al., J. Am. Chem. Soc., 1975, 97, 6584, Bioorg. Chem. 1979, 8, 351), and J. C. S. Perkin I 538 (1981)); (2) a second group of solid supports is based on silica-containing particles such as porous glass beads and silica gel. One example is the reaction product of trichloro-[3-(4-chloromethyl)phenyl]propylsilane and porous glass beads (see Parr and Grohmann, Angew. Chem. Internal. Ed. 1972, 11, 314) sold under the trademark "PORASIL E" by Waters Associates, Framingham, Mass., USA. Similarly, a mono ester of 1,4-dihydroxymethylbenzene and silica (sold under the trademark "BIOPAK" by Waters Associates) has been reported to be useful (see Bayer and Jung, Tetrahedron Lett., 1970, 4503); (3) a third general type of useful solid supports can be termed composites in that they contain two major ingredients: a resin and another material that is also substantially inert to the organic synthesis reaction conditions employed. A preferred support of this type is described in U.S. Pat. No. 5,235,028 which is herein incorporated by reference. One exemplary composite (see Scott, et al., J. Chrom. Sci., 1971, 9, 577) utilized glass particles coated with a hydrophobic, cross-linked styrene polymer containing reactive chloromethyl groups, and was supplied by Northgate Laboratories, Inc., of Hamden, Conn., USA. Another exemplary composite contains a core of fluorinated ethylene polymer onto which has been grafted polystyrene (see Kent and Merrifield, Israel J. Chem. 1978, 17, 243) and van Rietschoten in "Peptides 1974," Y. Wolman, Ed., Wiley and Sons, New York, 1975, pp. 113-116); and (4) continguous solid supports other than PEPS, such as cotton sheets (Lebl and Eichler, Peptide Res. 1989, 2, 232) and hydroxypropylacrylate-coated polypropylene membranes (Daniels, et al., Tetrahedron Lett., 1989, 4345), are suited for PNA synthesis as well.
While the solid-phase technique is presently preferred in the context of PNA synthesis, other methodologies or combinations thereof, for example, in combination with the solid-phase technique, apply as well: (1) the classical solution-phase methods for peptide synthesis (e.g., Bodanszky, "Principles of Peptide Synthesis," Springer-Verlag, Berlin-New York 1984), either by stepwise assembly or by segment/fragment condensation, are of particular relevance when considering especially large scale productions (gram, kilogram, and even tons) of PNA compounds; (2) the so-called "liquid-phase" strategy, which utilizes soluble polymeric supports such as linear polystyrene (Shemyakin, et al., Tetrahedron Lett., 1965, 2323) and polyethylene glycol (PEG) (Mutter and Bayer, Angew. Chem., Int. Ed. Engl., 1974, 13, 88), is useful; (3) random polymerization (see, e.g., Odian, "Principles of Polymerization," McGraw-Hill, New York (1970)) yielding mixtures of many molecular weights ("polydisperse") peptide or PNA molecules are particularly relevant for purposes such as screening for antiviral effects; (4) a technique based on the use of polymer-supported amino acid active esters (Fridkin, et al., J. Am. Chem. Soc., 1965, 87, 4646), sometimes referred to as "inverse Merrifield synthesis" or "polymeric reagent synthesis," offers the advantage of isolation and purification of intermediate products, and may thus provide a particularly suitable method for the synthesis of medium-sized, optionally protected, PNA molecules, that can subsequently be used for fragment condensation into larger PNA molecules; (5) it is envisaged that PNA molecules may be assembled enzymatically by enzymes such as proteases or derivatives thereof with novel specificities (obtained, for example, by artificial means such as protein engineering), and one also can envision the development of "PNA ligases" for the condensation of a number of PNA fragments into very large PNA molecules; and (6) since antibodies can be gernated to virtually any molecule of interest, the recently developed catalytic antibodies (abzymes), discovered simultaneously by the groups of Lerner (Tramantano, et al., Science, 1986, 234, 1566) and of Schultz (Pollack, et al., Science, 1986, 234, 1570), also should be considered as potential candidates for assembling PNA molecules. There has been considerable success in producing abzymes catalyzing acyl-transfer reactions (see for example Shokat, et al., Nature, 1989, 338, 269 and references therein). Finally, completely artificial enzymes, very recently pioneered by Stewart's group (Hahn, et al., Science, 1990, 248, 1544), may be developed to suit PNA synthesis. The design of generally applicable enzymes, ligases, and catalytic antibodies, capable of mediating specific coupling reactions, should be more readily achieved for PNA synthesis than for "normal" peptide synthesis since PNA molecules will often be comprised of only four different amino acids (one for each of the four native nucleobases) as compared to the twenty naturally occurring (proteinogenic) amino acids constituting peptides. In conclusion, no single strategy may be wholly suitable for the synthesis of a specific PNA molecule, and therefore, sometimes a combination of methods may work best.